The invention relates to biologically active molecules for influencing virus-, bacteria-, parasite-infected cells and/or tumor cells and a method for the use thereof. The aim of the invention is to influence virus-, bacteria-, parasite-infected cells and/or tumor cells physiologically, by means of one or more target proteases, even in the case of mutations of the target proteases.
The suggested biologically active molecules and the use thereof can be applied, in particular, for fighting and inhibiting the growth of abnormal cells, for example in tumor therapy and the treatment of virus infections, bacterial infections or infections with parasites.
The use of protease inhibitors in fighting virus infections was already described and has been applied for many years (for example EP 0691345 A3; U.S. Pat. No. 5,196,438 A; U.S. Pat. No. 5,541,206 A; U.S. Pat. No. 5,413,999 A; U.S. Pat. No. 5,484,926 A; U.S. Pat. No. 5,585,397 A).
The inhibition of the gene expression by introduction of short (19-23 bp), double-stranded RNA molecules (siRNA) or PNA molecules into eukaryotic cells, which is specific for a sequence segment of the mRNA of a target gene, was also described already (Elbashir S M et al.: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature, 2001 May 24, 411 (6836), 494-8; Liu Y et al.: Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids, Biochemistry, 2004 Feb. 24, 43(7), 1921-7; U.S. Pat. No. 5,898,031 A; U.S. Pat. No. 7,056,704 B2).
By means of such molecules, the reading of a gene and the production of an mRNA are not inhibited but in case of siRNA, a cell-intrinsic mechanism is initiated which degrades the target mRNA. Finally, as described earlier, the formation of a specific protein is suppressed without affecting the expression of further genes (post-transcriptional gene silencing).
For the suppression of the expression of a gene, siRNA and PNA molecules can be introduced directly into the cell by means of transfection reagents and electroporation (Zhang M et al.: Downregulation enhanced green fluorescence protein gene expression by RNA interference in mammalian cells, RNA Biol. 2004 May, 1(1), 74-7; Gilmore I R et al.: Delivery strategies for siRNA-mediated gene silencing, Epub 2004 May 22, Curr. Drug Deliv. 2006 April, 3(2), 147-5; U.S. Pat. No. 6,506,559 B1).
In this context, it is disadvantageous that siRNA is relatively instable, which can be improved by chemical modifications (U.S. Pat. No. 6,107,094 A).
Using biologically active molecules is particularly problematic for an application in vivo. For such application, possible methods were developed, for example stabilising siRNA molecules to reduce degradation (Morrissey et. al.: “Chemical Modifications of Synthetic siRNA”, Pharmaceutical Discovery, May 1, 2005), and transfection reagents, for example nanoparticles, in vivo-jetPEI™, were developed, which introduce siRNA into cells also in vivo (Vernejoul et al.: Antitumor effect of in vivo somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic cancer models, Cancer Research 62, 2002, 6124-31; Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A: RNAi-mediated gene-targeting through systemic application of poylethylenimine (PEI)-complexed siRNA in vivo, Gene Ther 12(5), 2005, 461-6).
Also, methods were developed wherein cells of a target tissue are increasingly transfected with siRNA in vivo (Ikeda et al.: “Ligand-Targeted Delivery of Therapeutic siRNA”, Pharmaceutical Research, Vol. 23, No. 8, August 2006).
The administration of biologically active substances in vivo, however, is often problematic due to their systemic effect. The selective introduction of these substances into target cells does not take place in a sufficiently specific manner. This is disadvantageous, in particular with siRNA, PNA and RNA molecules which are to be effective in a selective manner and in target cells exclusively. By means of tissue- and cell-specifically marked transfection reagents (e.g. antibody/antigen-marked nanoparticles, TAT protein flanking, among others), no sufficient cell specificity is achieved. Consequently, mistransfections take place.
For the compensation of said mistransfections, a mechanism is known where the biological effect of siRNA, PNA and RNA molecules is inhibited by binding of peptides and these peptides are cleaved by means of target cell active enzymes for activating of siRNA, PNA and RNA in the target cells (WO 2008098569 A2).
A general problem in the application of protease inhibitors for the inhibition of infectivity or replication of viruses, bacteria or parasites and the growth of tumors is that the viral, bacterial, parasite or tumor-specific enzymes are modified slightly in a very rapid manner, for example by mutations, and thus the applied inhibitors have no effect any longer. In this way, viruses, bacteria, parasites or tumor cells can proliferate again despite inhibitors applied.
Due to the rapid mutation rate of the genetic material in virus-, bacteria- or parasite-infected cells or tumor cells, the use of siRNA, PNA and RNA molecules per se is not considered appropriate or sufficiently efficient, since modifications of the mRNA target sequence of the siRNA, PNA or RNA can also inhibit the intended application effect of the molecules used.